Volume 98, Issue 1, Pages 115-124 (July 1999) Mechanisms Controlling Mitochondrial Biogenesis and Respiration through the Thermogenic Coactivator PGC-1  Zhidan Wu, Pere Puigserver, Ulf Andersson, Chenyu Zhang, Guillaume Adelmant, Vamsi Mootha, Amy Troy, Saverio Cinti, Bradford Lowell, Richard C. Scarpulla, Bruce M. Spiegelman  Cell  Volume 98, Issue 1, Pages 115-124 (July 1999) DOI: 10.1016/S0092-8674(00)80611-X Copyright © 1999 Cell Press Terms and Conditions

Figure 1 Stimulation of Oxygen Consumption by PGC-1 (A) Model illustrating the two predominant modes of respiration in mitochondria: oxidative phosphorylation and uncoupling. Continued oxygen consumption by mitochondria requires that protons reenter the mitochondrial matrix. This can occur via ATP synthase, if ADP is available, or proton leak, such as that mediated by UCPs. These two pathways can be distinguished using either oligomycin, an inhibitor for F1/F0 ATP synthetase, or FCCP, a chemical uncoupler. (B) PGC-1 increases both oxidative phosphorylation-linked and uncoupling-linked respiration in differentiated myotubes. C2C12 cells infected with virus containing an empty vector or an expression vector for PGC-1 were differentiated into myotubes. At day 5 postconfluence, the cells were gently detached from the dishes and transferred to a chamber for measurements of oxygen consumption. The concentration of FCCP and oligomycin were 2 μM and 2.5 μg/ml, respectively. Each measurement was performed in triplicate and normalized to the protein content of the cells. Cell 1999 98, 115-124DOI: (10.1016/S0092-8674(00)80611-X) Copyright © 1999 Cell Press Terms and Conditions

Figure 2 PGC-1 Increases Expression of Genes of the Mitochondrial Respiratory Chain (A) RNA analysis of PGC-1-expressing cells. Myoblasts expressing PGC-1 and the control cells were induced to differentiation and were then treated with various stimuli, including 100 nM T3 (24 hr) and 1 mM 8-bromo-cAMP (A) (6 hr). Total RNA was extracted and subjected to Northern blot analysis. Probes used for hybridization were PGC-1, β-ATP synthetase, COXII and IV, CytC, and myogenin. A cDNA encoding a ribosomal protein, 36B4, was also used as a control for loading equivalence of RNA. (B) Protein analysis of PGC-1-expressing cells. Total proteins were extracted from the cells at confluence (myoblasts) or day 5 postconfluence (myotubes) and were subjected to Western blot analysis. Cell 1999 98, 115-124DOI: (10.1016/S0092-8674(00)80611-X) Copyright © 1999 Cell Press Terms and Conditions

Figure 3 Cell Type–Selective Induction of UCPs through PGC-1 C2C12 and 3T3-F442A cells expressing PGC-1 and their controls were induced to differentiation as described in Experimental Procedures. The C2C12 cells were treated with various stimuli, including 1 mM 8-bromo-cAMP (A, 6 hr), 100 nM T3 (24 hr), or 10 μM troglitazone (Tro, 24 hr). 3T3-F442A adipocytes were treated with 100 nM T3 (18 hr) following with 1 μM 9-cis retinoic acid and 1 mM 8-bromo-cAMP for an additional 6 hr prior to RNA extraction. Total RNA was extracted and subjected to Northern blot analysis using 32P-labeled cDNAs encoding PGC-1, UCP-1, UCP-2, UCP-3, myogenin, and 36B4. Cell 1999 98, 115-124DOI: (10.1016/S0092-8674(00)80611-X) Copyright © 1999 Cell Press Terms and Conditions

Figure 4 PGC-1 Stimulates Mitochondrial DNA Replication and Biogenesis (A) Southern blot analysis of mitochondrial and genomic DNA. Total cellular DNA was isolated from C2C12 cells expressing PGC-1 and their controls, in both the myoblast and myotube states. Ten micrograms of DNA was digested with NcoI and subjected to Southern blot analysis using a cDNA for COX II as a probe for mtDNA. The blot was then stripped and hybridized to a cDNA for 36B4, a nuclearly encoded gene. (B) Transmission electron microscopy of PGC-1 expressing myoblasts and control cells. The magnification is 8750×. Cell 1999 98, 115-124DOI: (10.1016/S0092-8674(00)80611-X) Copyright © 1999 Cell Press Terms and Conditions

Figure 5 PGC-1 Controls Amount and Activity of Transcriptional Regulators of Mitochondrial Genes (A) PGC-1 increases the activity of the mtTFA promoter. BALB/c 3T3 cells were transfected with 0.25 μg of CMV/β-gal, 0.5 μg of the indicated mtTFA promoter luciferase reporter construct, and 4 μg of pSV/SPORT or pSV/SPORT-PGC-1. Luciferase activities were normalized to a cotransfected β-gal control. The data represent means of three independent experiments, each performed in duplicate. (B) PGC-1 induces mRNA for the NRFs and mtTFA. C2C12 cells expressing PGC-1 and the control cells were grown to confluence (day 0) or induced to differentiate for 5 days. Total RNA was extracted and 20 μg of each sample was subjected to Northern blot analysis. Probes used in hybridization were cDNAs for human NRF-1, NRF-2α, and mouse mtTFA. The same blot was also hybridized to cDNA for myogenin and 36B4. (C) Coactivation of NRF-1 by PGC-1. BALB 3T3 cells were transfected with 1 μg of a luciferase reporter gene containing 4× NRF-1-binding site, 0.5 μg of pGS5- NRF-1 expression plasmid, and the indicated amount of pSV/SPORT-PGC-1. Luciferase activities were normalized to a cotransfected β-gal control. The data represent the means of two independent experiments performed in triplicate. Cell 1999 98, 115-124DOI: (10.1016/S0092-8674(00)80611-X) Copyright © 1999 Cell Press Terms and Conditions

Figure 6 Physical Interaction of PGC-1 with NRF-1 (A) Interaction between PGC-1 and NRF-1 in cells. Vectors expressing the GAL4-DBD, GAL4-DBD-PGC-1, and NRF-1 were transfected in COS cells. Cells were harvested 48 hr after transfection and subjected to immunoprecipitation and Western blot analysis. (B) Mapping of the domain of PGC-1 interacting with NRF-1 in vitro. Portions of PGC-1 fused to GST were bound onto beads and incubated with in vitro translated [35S]-PPARγ or [35S]-NRF-1. The beads were then washed, and the bound proteins were eluted and analyzed by autoradiography after SDS–PAGE. The retained PPARγ and NRF-1 were quantified using a PhosphoImager, and the results shown were expressed as percentages of the inputs. (C) Mapping of the domain of NRF-1 interacting with PGC-1 in vitro. PGC-1 deletions fused to GST were bound to beads and incubated with different [35S]-NRF-1 fragments. The bound proteins were washed, eluted, and analyzed by autoradiography after SDS–PAGE. (D) Schematic representation of key interaction domains on PGC-1 and NRF-1. Note that there is overlap in the domains of PGC-1 that interact with NRF-1 and PPARγ. Cell 1999 98, 115-124DOI: (10.1016/S0092-8674(00)80611-X) Copyright © 1999 Cell Press Terms and Conditions

Figure 7 A Dominant-Negative Allele of NRF-1 Blocks the Effect of PGC-1 on Mitochondrial Biogenesis C2C12 myoblasts were transfected with an IRSE bicistronic expression vector encoding both PGC-1 and GFP (PGC1-IRSE2-EGFP) along with either empty vector (pSG5) (B) or the dominant-negative NRF-1, pSG5-NRF-1/DN (C). The control cells (A) were transfected with pSG5 and the GFP vector (pIRSE2-EGFP). Cells were stained with MitoTracker Red CMXRos 60 hr posttransfection. The GFP-positive cells were sorted, and the MitoTracker fluorescence of these cells was analyzed by flow cytometry. The mean of the MitoTracker fluorescence was 115.6 in (A), 219.2 in (B), and 138.0 in (C). Cell 1999 98, 115-124DOI: (10.1016/S0092-8674(00)80611-X) Copyright © 1999 Cell Press Terms and Conditions

Figure 8 Pathway of Mitochondrial Biogenesis through PGC-1 Cold and high-calorie diet stimulate the sympathetic nervous system leading to the release of norepinephrine. NE triggers the activation of the β-adrenergic receptors (AR) resulting in the elevation of intracellular cAMP and inducing the expression of PGC-1. PGC-1 activates the expression of the subunits of respiratory chain and mtTFA through the induction of the expression of NRFs and the coactivation of NRF-1-mediated transcription. mtTFA subsequently translocates into mitochondrion and directly activates the transcription and replication of mtDNA. Cell 1999 98, 115-124DOI: (10.1016/S0092-8674(00)80611-X) Copyright © 1999 Cell Press Terms and Conditions